Liquid chromatography is commonly used for analyzing the composition of chemical compounds whereby solutions containing the compounds to be analyzed are forced under pressure through a liquid chromatography (LC) column. The LC column is specifically constructed to interact with the pressurized solution and affect the fluid flow rate in a way that is characteristic of the composition of chemicals in the solution. Components are thereby separated in the solution according to their chemical composition and corresponding flow rate. Eluent from the LC column is typically in a highly diluted liquid phase. Separated components are manifested as “chromatographic peaks” on detector apparatus.
In liquid chromatography analysis, the choice of an appropriate separation strategy, including the combined implementation of hardware, software, and chemistry, results in the separation of an injected sample into separate components, which elute from the column in reasonably distinct zones or “bands”. As these bands pass through a detector, a detector output, usually in the form of an electrical signal, is produced. The pattern of analyte concentration within the eluting bands, which can be represented by means of a time-varying electrical signal, gives rise to the nomenclature “chromatographic peak” (or “peak”). Peaks may be characterized with respect to their “retention time,” which is the time at which the center of the band transits a detector. In many applications, the retention time of a peak is used to infer the identity of the eluting analyte based upon related analyses with standards and calibrants. The retention time for a peak is strongly influenced by the mobile phase composition of the analyte and by the accumulated volume of mobile phase which has passed through the LC column.
It is often desirable to separate and collect specific component(s) from the HPLC separation of a complex mixture for further testing and evaluation. For example, samples of a pure component may be needed to evaluate the biological activity or other properties of a candidate drug molecule.
Conventional methods for physically collecting a purified sample from an eluent stream employ a fraction collector which is capable of diverting a portion or “fraction” of the stream into a collection vessel at a specified time. The specified time for opening the fraction collector ideally coincides with the arrival at the fraction collector of concentrated components of the eluent stream.
As is known in the art, timing of a fraction collector in an LC eluent stream can be controlled by installing a triggering detector which initiates a timer upon detection of a particular component concentration (peak). The fraction collector is triggered after a delay time elapses.
Any detector which senses a peak before it reaches the fraction collector can be used to trigger fraction collection. UV detectors are commonly used as triggering detectors in UV-directed purification or fraction collection systems. Mass spectrometers are commonly used as triggering detectors in mass-directed purification or fraction collection systems. These detectors can recognize a signature peak representing the presence of particular components and trigger the fraction collector to collect only desired components from the eluent stream. The triggering detector can be installed upstream of a fraction collector if it is a non-destructive detector such as a UV detector. However, the triggering detector is not necessarily installed directly upstream of a fraction collector. It can also be installed in one branch of a split flow wherein the other branch of the split flow is directed to the fraction collector. For example, if a destructive detector such as a mass spectrometer is used as a triggering detector, it must be installed in a separate branch of the eluent stream.
The delay time is the time between the appearance of a peak at the triggering detector and the arrival of the peak at the fraction collector. This time depends on the flow rate and fluid volume in the relevant connecting tubing. The delay time of an eluent stream can be determined by injecting a known calibrant such as a visible dye at an upstream location and recording the elapsed time between its detection by the triggering detector and its arrival at the fraction collector. One drawback of this calibration method is that the special calibrant dye must be injected whenever it is desired to check that the correct delay time is being used. For example, a change in flow rate for any reason will result in a change in delay time, which could go unnoticed.
Mass spectrometers are commonly used to analyze the composition of an eluent stream. U.S. Pat. No. 6,406,633 to Fischer et al. (“Fischer '633”) and U.S. Pat. No. 6,106,710 to Fischer et al. (“Fischer '710”) disclose a fraction collection system which directs a portion of an eluent stream from a liquid chromatography column to a mass spectrometer, and detects a desired component (peak) with the mass spectrometer. The apparatus of the Fischer disclosures is shown diagrammatically in FIG. 1. The mass spectrometer 52 triggers a delay timer for controlling the actuation of a fraction collector 54 in a separate branch of the stream. The eluent stream flows from a liquid chromatography column 51 to a splitter 57 which divides the eluent stream and directs one branch to a mass spectrometer 52 and another branch to a fraction collector 54. Even though the mass spectrometer 52 disclosed in Fischer '633 and Fischer '710 is disposed in a separate stream branch 53 rather than directly upstream of the fraction collector 54, the mass spectrometer 54 can be used to time the opening of the fraction collector 54 if the flow rates in each branch of the stream are related in a predictable way, and the peak is detected by the mass spectrometer before it reaches the fraction collector. A downstream detector 55 is disposed near the fraction collector 54. Detection of a peak at the downstream detector 55 allows the flow rate of the peak to be determined by measuring the elapsed time between detection of a sample upstream and its arrival at a downstream point near the fraction collector. The downstream detector 55 described in Fischer '633 and Fischer '710 is a non-destructive detector such as a UV detector.
To effect timing of the fraction collector 54 Fischer '633 discloses a delay time determined empirically by the injection of a calibrant in the eluent stream and timing the arrival of the calibrant at the mass spectrometer 52 in one branch of the stream. The arrival of the calibrant at downstream detector 55 proximate to fraction collector 54 in a separate branch of the stream is also timed. The delay between arrival of the calibrant at the mass spectrometer 52 and arrival of the calibrant at the downstream detector 55 provides flow rate information that can be used to time the opening of the fraction collector after a sample being analyzed is detected in the mass spectrometer.
Each of the various implementations described in Fischer '633 and Fischer '710 involves the use of a destructive detector such as a mass spectrometer to identify the presence of a substance in an eluent stream. The use of a destructive detector requires splitting the eluent stream into an analyzed stream flowing to the destructive detector and a collection stream flowing to the fraction collector. Fischer '633 and Fischer '710 also disclose various implementations of a third detector upstream from the fraction collector to better characterize the flow in each branch of the eluent stream in order to more accurately time the opening and closing of the fraction collector. The third detector is described in Fischer '633 and Fischer '710 as a non-destructive detector such as a UV detector.
The various implementations described in Fischer '633 and Fischer '710 require placement of a non-destructive detector near the fraction collector. Such placement is disadvantageous for use because typical fraction collectors use long robotic arms to dispense into collection vessels. If a detector must be located near a fraction collector dispensing head, only detectors suitable for mounting on a robotic arm can be used. This precludes use of standard HPLC detectors, for example a tunable UV detector, which can be used to detect a variety of components being separated, but are not suitable for mounting on a robotic arm.
No information regarding the accuracy of the fraction collector timing signal is provided by any apparatus described in Fischer '633 or Fischer '710. Furthermore, the various apparatus described in Fischer '633 and Fischer '710 do not provide confirmation that a desired component was successfully collected by a fraction collector. The delay time in Fisher '633 is determined using a calibrant. It is assumed to remain unchanged during sample collection, until such time as calibration is repeated.
Each of the various fraction collection methods described in Fischer '633 requires the use of a calibrant which is injected into the eluent stream for calibrating the delay period before the fraction collector is actuated. Persons skilled in the art will recognize that any calibrant may have different flow characteristics than the substance being analyzed. For example, a change in flow rate or split ratio could occur after calibration is performed. The calibrated delay period therefore may include errors that result in non-optimal collection of a desired substance at the fraction collector. Even small errors in timing can cause a fraction collector to miss much or all of a target substance.
None of the methods and apparatus heretofore known for controlling fraction collector timing in LC systems include a means to confirm the successful collection of a desired component in the eluent stream.